CN113308512A - Dry transplanting method for conidia of banana fusarium wilt - Google Patents
Dry transplanting method for conidia of banana fusarium wilt Download PDFInfo
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Abstract
A dry transplanting method for conidia of banana wilt pathogen, adhering spores from spore-forming colonies, directly transplanting and spreading the spores on an observation plate, wherein the dry transplanting operation steps of the spores are as follows: 1) preparing conidia for culture; 2) aseptic processing of the spore-removing cotton swab; 3) preparing an observation agar plate; 4) the dispersion of dense spores; 5) performing transfer printing on conidia; 6) and (5) culturing conidia. The invention has the advantages that: 1) the key working materials (conidia of the fusarium oxysporum f.sp.cubense) do not need to be prepared into spore suspension, so that the pollution risk is reduced. 2) The method can spread the conidia of the banana vascular wilt pathogen on a strip-shaped agar plate, observe the germination and growth activities of the conidia, improve the efficiency and effect of observation work, and facilitate the development of multi-factor action design and batch test comparison in the biological research of spore germination.
Description
Technical Field
The invention relates to a plant pathology technology, in particular to a dry transplanting method of conidia of banana fusarium wilt.
Background
The banana wilt is an important disease in the banana industry of China, can cause great loss to banana production, and can seriously damage the whole banana garden. The disease is caused by banana Fusarium oxysporum f.sp.cubense infection. Conidia are asexual propagules of banana vascular wilt and play an important role in disease circulation and disease prevalence of banana vascular wilt. Fully knowing the germination biology of conidia is crucial to effectively preventing and controlling the occurrence and prevalence of banana vascular wilt. In the germination biology of banana fusarium oxysporum propagules and related research work, conidia are often transplanted to a culture plate for observation culture, and the observation culture not only requires that the spores on the culture plate are in a proper dispersed state, but also requires that the number and distribution of the spores on each transplantation treatment plate are consistent. Only then is the relevant research task done with high quality.
The main procedure of the method is that firstly, a proper spore-producing culture medium is used for spore-producing culture of banana vascular wilt pathogens, then spores of the spore-producing culture colony are washed out by water and prepared into a spore suspension (spore liquid for short), then a liquid transfer device is used for quantitatively transferring the spore liquid to the surface of a culture medium observation flat plate, and a tool is used for coating and dispersing the spore liquid on the flat plate, and then test work such as related culture, observation and the like is carried out. The method seems to be simple, but the practical operation still has considerable difficulty and complexity, and is mainly shown in that in the operation of obtaining a key working material (conidia of banana fusarium wilt), filtration operation is often needed to remove hyphae in spore liquid, centrifugal precipitation operation is also needed to remove a culture medium, a culture metabolite and the like in the spore liquid, the operation processes have large pollution risks, in addition, the spore concentration of the spore liquid needs to be adjusted to a proper range, so that repeated blending and sampling are needed to a counting plate or a glass slide, and the counting needs to be observed and counted under a biological microscope, and the operation is complicated.
Disclosure of Invention
The invention aims to provide a dry transplanting method of conidia of fusarium oxysporum f.sp.cubense.
The technical scheme for solving the technical problems is as follows:
a dry transplanting method for conidia of banana wilt pathogen, adhering spores from spore-forming colonies, directly transplanting and spreading the spores on an observation plate, wherein the dry transplanting operation steps of the spores are as follows:
1. preparation of conidia for culture
And (3) carrying out sporulation culture on the banana fusarium oxysporum by using a potato sucrose agar culture medium until dense conidia are formed on sporulation colonies.
2. Aseptic processing of spore-removing swabs
Firstly, a marker pen is used for stippling and marking on a common cotton swab, then the cotton swab is placed into a culture dish, and the cotton swab is sterilized at high pressure and dried for later use.
3. Preparation of Observation agar plates
Preparing a spore observation plate by using a conventional agar culture medium, cutting the agar plate poured from the culture dish into a strip shape by using a sterilization blade, and picking and transferring the agar strip onto a sterilization glass slide to be used as a conidium observation plate.
4. Dispersion of dense spores
Taking the sterilized cotton swab prepared in the step 2, touching the spherical surface at the end of the cotton swab with the spore-producing bacterial colony prepared in the step 1, and adhering dense spores on the bacterial colony by the spherical surface contacting the bacterial colony; then transferring to a blank agar plate for smearing and spotting, and scattering dense spores adsorbed on the spherical surface of the cotton swab.
5. Transfer printing of conidia
And (4) orderly and continuously dotting the spherical surface of the cotton swab with the scattered spores in the step (4) at different positions of the spore observation agar plate surface prepared in the step (3) to obtain spore prints with consistent spore quantity and good spore dispersibility.
6. Culture treatment of conidia
And (5) placing the observation agar plate with the spore blot obtained in the step (5) into a culture dish with a moisturizing effect, culturing under a culture condition set by work, and observing and measuring the germination and growth characteristics of the conidia according to the work requirement.
THE ADVANTAGES OF THE PRESENT INVENTION
1) The key working materials (conidia of the fusarium oxysporum f.sp.cubense) do not need to be prepared into spore suspension, so that the pollution risk is reduced.
2) The method can spread the conidia of the banana vascular wilt pathogen on a strip-shaped agar plate, observe the germination and growth activities of the conidia, improve the efficiency and effect of observation work, and facilitate the development of multi-factor action design and batch test comparison in the biological research of spore germination.
Drawings
FIG. 1 shows that the end spherical surface of a cotton swab touches adhered spores once on a spore-forming colony of Fusarium oxysporum f.sp.cubense, and then is spotted on an agar plate surface for multiple times, wherein 4 blots obtain the number and the dispersion state of the spores. FIG. 1-1 shows the first spotting on the agar surface, FIG. 1-2 shows the 20 th spotting on the agar surface, FIG. 1-3 shows the 30 th spotting on the agar surface, and FIG. 1-4 shows the 50 th spotting on the agar surface. The black oval points in the figure are conidia of banana vascular wilt bacteria observed under a microscope. The black short bar at the lower part of the figure is 50 μm long
FIG. 2 shows that the end spherical surface of a cotton swab touches and adheres to conidia only once on a spore-forming colony of fusarium oxysporum f.sp.cubense, then is transferred to the flat surface of a potato sucrose agar culture medium poured from a 9cm flat dish, is continuously and orderly spotted with the spores 195 times, is transferred to a temperature of 28 ℃ for culturing for 35 hours, and releases scattered spores on each imprinted position to germinate and grow to form a colony. The order of the dotting is as follows: the head row is at the top and the tail row is at the bottom; each row runs from left to right. The growth amounts of the adjacent multiple imprinted colonies in the figure were consistent. In the figure, the colony marked a is the colony formed by growth of the 1 st blot, and the colony marked b is the colony formed by growth of the 195 th blot.
FIG. 3 is a diagram showing the state of the germination and growth of conidia of Banana Fusarium oxysporum strain Foc4-2 after the conidia are transplanted and spread from spore-forming colonies onto the surface of an observation plate, the state of the dispersion of the conidia, and the state of the germination and growth of the conidia after culturing at 28 ℃ for 13 hours by the method of the present invention. The black short bar at the lower part of the figure is 50 μm long
Detailed Description
In the daily work of banana vascular wilt research, the inventor uses the common cotton swab to transfer the end spherical surface to the flat surface for spot printing after the end spherical surface touches spore-forming bacteria of banana vascular wilt, and conidia adhered to the spherical surface of the cotton swab can be released and scattered on the flat surface of agar. Surprisingly, the surface of the cotton swab contacts only one spore-forming colony, the spores can be continuously spotted on an agar plate for more than 170 times, and the spores in the blot are observed, so that the dispersion of the spores in most blots is good, as shown in figure 1, and the number of the spores in adjacent blots is consistent. The culture plate with 195 times of ordered continuous spot-printed spores is placed at the temperature of 28 ℃ for culturing for 35 hours, spores of all blots germinate and grow to form visible colonies, and the colony growth amount of a plurality of adjacent blots is consistent, which indicates that the spore number of each blot shows a process of gradually decreasing from one blot to another, and is shown in fig. 2. The result shows that a plurality of spore blots with consistent spore quantity and good dispersibility can be printed on the culture plate only by touching the spore-forming colonies once, and the effect is exactly the technical effect required by conidium transplantation in a plurality of researches on the biology of the fusarium oxysporum f.sp. The present invention can achieve such technical effects by performing the following specific steps.
1. Preparation of conidia for culture
The conventional spore-producing culture of the banana vascular wilt germs can be carried out by using a spore-producing culture medium of the banana vascular wilt germs such as potato sucrose agar and the like until a large number of conidia are formed on a spore-producing substrate, usually, the culture is carried out for 7-10 days at the temperature of 28 ℃, and a large number of dense conidia can be formed on a flat bacterial colony.
2. Aseptic processing of spore-removing swabs
The common cotton swab is used as a transplanting tool for conidia of banana wilt pathogens, the circumferential direction and the direction of a cotton ball at the end of the cotton swab are usually not easy to identify, a marker pen can be used for stippling a mark near the cotton ball to facilitate the identification of the direction of the cotton ball, the marked cotton swab is put into a culture dish or other small containers, and the cotton swab is sterilized at high pressure and dried for later use.
3. Preparation of Observation agar plates
Spore-observing plates are prepared using a conventional medium containing agar, such as potato agar. The culture medium is heated and melted, poured into a flat-bottom culture dish to be made into a common flat plate, after the flat plate is condensed, the flat plate of the culture medium is cut into slender strips by a sterilization operating blade, and the strip-shaped flat plate of the culture medium is picked up by the blade and transferred onto a sterilization glass slide to be used as a flat plate for observing and measuring the germination biology of the conidia of the fusarium oxysporum f.sp.cubense. The length of the observation plate can be consistent with the length of the glass slide, and the width of the observation plate can be slightly wider than a cotton ball of the cotton swab.
4. Dispersion of dense spores
Taking out the sterilized cotton swab with the orientation marks prepared in the step 2, touching the spherical surface of the end of the cotton swab on the surface of the spore-forming colony prepared in the step 1, wherein a large number of dense conidia can be adhered to the spherical surface of the cotton swab, transferring the cotton swab to the surface of a common blank agar plate, according to the orientation marks near the cotton ball, touching the spherical surface of the colony by the cotton swab to be aligned with the agar plate, performing a slight smearing operation on the surface of the plate, wherein the dense spores adsorbed on the spherical surface of the cotton swab can quickly release the dispersion, usually, the plate surface is slightly smeared for 2-3 times, the dispersion state of the spores left on the spherical surface of the cotton swab meets the working requirements, and after the smearing operation, the spherical surface of the cotton swab is printed on the agar surface in a spot printing mode, and then the blots of the spores are transferred to a microscope for inspection, so that the dispersion degree or the dispersion distance of the spores meets the working requirements. It should be noted that the conidia of banana wilt pathogen are easy to be inactivated in dry environment for a long time, and in order to avoid the inactivation of the spores on the spherical surface of the cotton swab, the cotton swab with the adhered spores can be placed in a moisture-preserving container for protection during microscopic examination.
5. Transfer printing of conidia
And 4, after confirming that the state of the spores left on the spherical surface of the cotton swab meets the working requirement, transferring the cotton swab to the spore observation flat plate surface prepared in the step 3, aligning the spherical surface with the spores to different positions of the observation flat plate surface according to the direction mark of the cotton swab, sequentially and continuously dotting, sequentially releasing the spores on the spherical surface to scatter at each dotting position of the observation flat plate surface, determining the number of times of dotting according to the working requirement, and obtaining the spore prints with consistent number of the spores and good dispersibility after finishing the dotting.
6. Culture treatment of conidia
And (3) placing the observation agar plate carrying the conidium blot obtained in the step (5) into a culture dish or other moisture-preserving small containers filled with wet paper or wet gauze in the bottom of the dish, culturing the conidia and the moisture-preserving small containers under the culture condition set by the work, and observing and measuring the germination and growth characteristics of the conidia according to the work requirement.
Example 1
By adopting the dry-type transplanting method for the conidia of the banana fusarium oxysporum, the conidia of the strain Foc4-2 are transplanted to a spore observation agar plate from a spore-producing bacterial colony on a potato sucrose culture medium, and the result shows that the dispersion of the conidia on the surface of the agar plate is good; after the observation agar plate is cultured for 13 hours at the temperature of 28 ℃, the observation agar plate is taken out and observed under a microscope to observe each dot-printed blot, spores and sprouted germ tubes in each blot can be clearly identified, as shown in figure 3, 150 spores in one blot are randomly observed, and the germination rate of the normally germinated spores is 91%.
Claims (1)
1. A dry transplanting method of conidia of banana fusarium wilt is characterized in that spores are adhered to spore-producing colonies and are directly transplanted and spread on an observation plate, and the dry transplanting operation steps of the spores are as follows:
1) preparation of conidia culture: performing spore-producing culture of banana fusarium oxysporum by using a potato sucrose agar culture medium until dense conidia are formed on spore-producing colonies;
2) aseptic processing of the spore-removing cotton swab: firstly, a marking pen is used for stippling and marking on a common cotton swab, then the cotton swab is placed into a culture dish, and the cotton swab is sterilized at high pressure and dried for later use conventionally;
3) preparation of observation agar plates: preparing a spore observation plate by using a conventional agar culture medium, cutting the agar plate poured from a culture dish into a strip shape by using a sterilization blade, and picking and transferring the agar strip onto a sterilization glass slide to be used as a conidium observation plate;
4) and (3) dense spore dispersion: taking the sterilized cotton swab prepared in the step 2), touching the spherical surface of the end part of the cotton swab with the spore-producing bacterial colony prepared in the step 1), and adhering dense spores on the bacterial colony by the spherical surface contacting the bacterial colony; then transferring the mixture to a blank agar plate for smearing and dotting, and scattering dense spores adsorbed on the spherical surface of a cotton swab;
5) transfer printing of conidia: orderly and continuously dotting the spherical surface of the cotton swab on which the spores are scattered in the step 4) at different positions of the spore observation agar plate surface prepared in the step 3) to obtain spore imprints with consistent spore quantity and good spore dispersibility;
6) and (3) culture treatment of conidia: placing the observation agar plate loaded with the spore blot obtained in the step 5) into a culture dish with a moisturizing effect, culturing under a culture condition set by work, and observing and determining the germination and growth characteristics of the conidia according to the work requirement.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113278578A (en) * | 2021-06-15 | 2021-08-20 | 广西大学 | Transplanting method of rice blast bacterium conidia |
| CN113308432A (en) * | 2021-06-15 | 2021-08-27 | 广西大学 | Transfer and dispersion method of ustilaginoidea virens thin-walled conidia |
| CN113308431A (en) * | 2021-06-15 | 2021-08-27 | 广西大学 | Transplanting and dispersing method for botrytis cinerea conidia |
| CN113308430A (en) * | 2021-06-15 | 2021-08-27 | 广西大学 | Dispersion culture method of banana colletotrichum gloeosporioides conidia |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113278578A (en) * | 2021-06-15 | 2021-08-20 | 广西大学 | Transplanting method of rice blast bacterium conidia |
| CN113308432A (en) * | 2021-06-15 | 2021-08-27 | 广西大学 | Transfer and dispersion method of ustilaginoidea virens thin-walled conidia |
| CN113308431A (en) * | 2021-06-15 | 2021-08-27 | 广西大学 | Transplanting and dispersing method for botrytis cinerea conidia |
| CN113308430A (en) * | 2021-06-15 | 2021-08-27 | 广西大学 | Dispersion culture method of banana colletotrichum gloeosporioides conidia |
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